Efficacy of Binary Media and Gold Catalyst for the Synthesis of a Conjugates with Cyclohexyl-Tetrazole-Alkyloxyphenyl-Benzenamine through Ugi 4-CC Reactions: Cytotoxic and Single-Crystal Studies

Abstract With the critical increase in patients suffering from various types of malignant growth, it is obvious that quick steps in the development of novel and viable agents should be taken. In the current study, we focused on a novel synthetic approach for the synthesis of tetrazole derivatives using Ugi-4CR by employing a gold catalyst in water: methanol media. Gold’s adjustable nature, unique characteristics, and mild reaction conditions were utilized to generate a quadrupole adduct containing a cyclohexyl system, benzamine, terminal alkyne bearing aryl and a medicinally efficient tetrazole fragment. Along with the routine characteristic techniques (IR, MS, NMR), single crystal XRD was imparted for the assumption of the spatial arrangement of the desired adduct (5a-5j). Out of 10 selected scaffolds for the NCI 60 cell lines study, 5b was shown their highest potency in the single-dose assay (10−4 M) against CAKI-1 (GI50= −4.66, Renal cancer) and 5e were found to be most active in Leukemia (MOLT-4, GI50= −5.37) cancers than the rest of synthesized molecules.


Introduction
5][6] One of the solutions to expedite the discovery of cost-effective drugs is to generate a library of compounds via an efficient and rapid synthetic method that can provide chemically diverse starting points without following a lengthy sequence of reactions.8][9] Among various traditional MCRs, Ugi stand for many decades to serve and given the number of lead molecules in the current drug discovery process due to its ease of reaction, moderate reaction conditions, stereospecific product formation, wide availability of the reactants and higher yield. 10ver the past few years, interest in tetrazole chemistry has been increasing rapidly because of its wide range of applications in several fields such as medicine, 11 biochemistry, 12 pharmacology, 13 and in the industry as materials, e.g., in photography, 14 imaging chemicals, 15 and military, 16 mainly due to the role played by this heterocyclic functionality in medicinal chemistry as these offer a more favorable pharmacokinetic profile and a metabolically stable surrogate for carboxylic acid functionalities. 17Tetrazoles are such a type of heterocyclic that has excellent therapeutic application in the field of drugs and also has a minimal side effects on the host (Figure 1).Some of these lead molecules exhibit numerous kinds of biological belongings; Antimicrobial, [18][19] anticonvulsant, [20][21] anticancer, [22][23] analgesic, 24 antiulcer 25 anti-allergyic 26 and antibiotics. 27][34] Interestingly, gold catalysts have not only emerged as superior contenders for catalyzing the reactions of alkynes, alkenes, and allenes but also more intriguingly been found to provide divergent reaction pathways 35 compared to other p-acid catalysts such as Ag, Pt, Pd, Rh, Cu, In, Sc, Hg, Zn, etc.The unique reactivity exhibited by gold catalysts is due to their strong p-acidic nature and their potential to stabilize cationic intermediates generated during a reaction distinctly. 368][39] In continuation of our work to generate a novel and best anticancer scaffold, we described the gold-catalyzed Ugi 4-component optimized process for hybrid generation by clubbing of four diverse fragments, i.e., cyclohexyl, tetrazole, alkyloxyphenyl, and benzenamine.With the aiming of showing the best cytotoxic potency, derived adducts were screened for NCI 60 cell lines and correlates the active scaffolds with their structural properties.

Materials and methods
Chemicals and solvents were purchased from Sigma-Aldrich Chemical Co., Merck chemical, Finar and Spectrochem Ltd.The entire chemicals were used without further purification unless otherwise noted.Thin-layer chromatography was accomplished on 0.2 mm precoated plates of silica gel G60 F254 (Merck).Visualization was made under UV light (254 and 365 nm) or with an iodine chamber.IR spectra were recorded on an IR Affinity-1S spectrophotometer (Shimadzu). 1 H (400 MHz), 13 C (101.1 MHz) NMR spectra were recorded on a Bruker AVANCE II spectrometer in CDCl 3 .Chemical shifts are expressed in d ppm downfield from TMS as an internal standard.Mass spectra were determined using a direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu).BUCHI rotary evaporator was used for the isolation of products and recovery of solvents.Melting points were measured in open capillaries and are uncorrected.Column chromatography was carried out using Silica Gel 60-120 Mesh (Sigma-Aldrich) at a standard flow rate (ambient temperature and pressure).

Preparation of single crystals of N-((1-cyclohexyl-1H-tetrazol-5-yl)(2-(prop-2-ynyloxy)phenyl)methyl)benzenamine (5a).
Purely synthesized compound 5a (LC-MS purity: 95%) (1.0 g) was taken in Chloroform: Methanol (1:1) and heated up to 50-60 C for 10-15 minutes till it dissolved completely (the super saturated solution was made).Activated charcoal was added and further, it was heated up to 50-60 C for 5 minutes.The hot solution was filtered through wattmann 41 filter paper, followed by a hyflow (Celite) bed under a high vacuum.The solution was allowed to cool gradually and kept in a stoppered conical flask.The crystals were grown up in a few months (48 days).

Chemistry
In our previous reports, we had discussed the role of alicyclic, alicyclic with foreign elements, heterocyclic and a variety isocyanide based Ugi reactions to identify the active scaffolds which may serve as a novel drug in medicinal chemistry [39][40][41] .
In this study, we started our study using 2-(prop-2-yn-1-yloxy)benzaldehyde 1 (1 mmol), substituted amine 2 (1 mmol), Cyclohexyl isocyanide 3 (1.2mmol) and TMSN 3 4 (1.5 mmol) as the model components to evaluate reaction conditions (Table 1) (Scheme 1).Inspired by previous literature reports with transition metals as initiators, we tested the reactions in original catalyst-free versions upon mixing various model components as described.When we optimized the reaction condition without catalyst, it was found that the yield was meager with varying side products as well.
The reaction condition was then optimized based on the yield of the desired product for a model reaction designated as 5a (Scheme 1, R 5 H).As shown in Table 1, it was found that the yield was inadequate without a catalyst (10%, Table 1, entry 1).The yield was improved but still lower than 50% in the presence of some catalysts like Iron, copper, Silver, Iridium, palladium, FeCl 3 DCM 20 RT Trace a Each catalyst was optimized using a 5 mol% amount and beyond that, no changes were observed in the yield of the product.
b All the polar solvents were used after drying in the desiccator immediately.c Conversation was checked using LC-MS analysis.

POLYCYCLIC AROMATIC COMPOUNDS
and BINOL (30% to 50%, Table 1, entries 8-15).The switch of selectivity for the formation of the desired product depending on the gold catalyst was analyzed based on the high degree of conversion to desired adducts.The application of 5 mol% of AuCl or AuCl 3 as a catalyst in MeOH produced Tetrazole only in 25% and 35% conversion, respectively, while no reaction was observed with Au(PPh 3 )Cl (Table 1, entries 1-4).When the gold catalyst was added, the yield varied due to its chemical stability and highly active and selective domino coupling and condensation reaction.Later stage the use of cationic Gold i.e., Au(PPh 3 )OTf (5 mol%) at varying temperatures and solvents showed unexpected changes in the conversation ratio.It was shown major effect on single and dual solvent needs and at RT, the conversation was only 47% using pure methanol as media while using dual solvent (Me-OH: Water) in equal volume enhances almost double (93%) conversation at elevated temperature (50 C) (table 1, entries 5 and 6).The switch of selectivity for the formation of tetrazole depends on the use of cationic Gold (I) (Table 1, entry 5) or an AuCl/AuCl 3 catalyst (Table 1, entries 1 and 2) encouraged us to optimize the conditions further.The application of Au(PPh 3 )SbF 6 (5 mol%) at RT produced only 30% of conversion while employing AgOTf (5 mol%) solely at RT did not give any conversion.Interestingly upon heating at 50 C, AgOTf gave 50% 5a as the sole product in 24 h (Table 1, entries 8-9).Interestingly, PtCl 2 (5 mol %) under different solvent media did not show any conversion at RT, while upon heating at 50 C, 55% conversion was obtained.
The reaction media was screened subsequently.The results indicated that the reaction did not perform in pure water (Table 1, entry 4) because starting materials were poorly soluble in water.Methanol gave the best result in several pure organic solvents, but the yield of the product till low (Table 1, entry 6).Interestingly, when a 1:1 (v/v) mixture of CH 3 OH/H 2 O was used as a reaction medium, the yield of the product had a more increased (Table 1, entry 5, 93%).Water accelerated the reaction rate through hydrophobic aggregation, which effectively gathered the organic reagents and enabled them to interact with each other at a high concentration.And polar solvent could also stabilize the iminium ion of the reaction 42,43 .Above all, the optimum condition was using 5 mol% gold salt as a catalyst, 2 mL of the mixture of CH 3 OH and H 2 O (1:1) as solvent at 50 C for 3 h.
With the optimized condition in hand, we explore the substrate scope of this transformation (Table 2).Table 2 showed that the reaction could efficiently afford desired products in high yields (75 to 98%) with broad substrate scope.There was no stable relationship between the steric hindrance, electronic effect of substitution and the yield of products.The reaction was brutal when the substrate contained strong electron-withdrawing groups such as nitro because they may cause instability of the iminium ion intermediate.A variety of aromatic amines could yield relative product efficiently, which showed the high efficiency and universality of the catalyst.
The predicted structure of the compounds 5a has been confirmed by Single Crystal XRD and shown in Figures 2 and 3 with CCDC No. 2106364.

Cytotoxic study
The compounds (5a-5j) were screened for preliminary anticancer assay by National Cancer Institute (NCI), Bethesda, Maryland, the USA in an in vitro 60 human tumor cell lines panel, derived from nine neoplastic cancer types.The compounds (5a-5j) were submitted to NCI under the Developmental Therapeutic Program (DTP) which operates a tiered anticancer compound screening course for the benefit of the general research community to identify novel chemical leads and biological mechanisms.Structures of the compounds were selected for screening based on their ability to add diversity to the NCI small molecule compound collection.The process utilized 60 different human tumor cancers of the lung, colon, brain, ovary, breast, prostate and kidney which was aimed at showing selective growth inhibition or cell killing of particular tumor Table 2. Scope of the Ugi-4CR reaction under Gold (I) as a catalyst (5 mol%) a in the entire series of 5a-5j.cell lines by the specific compound.The screening begins with the evaluation of all selected compounds against these 60 cell lines at a single dose of 10 À5 M. In addition, the submission of compounds with drug-like properties utilizing the concept of privileged scaffold or structures based on the computer-aided design was preferred [44][45][46] .All synthesized molecules, (5a-5j) were selected for their anticancer screening for a single dose-response study.The output from the single-dose screen is reported as a mean graph and is available for analysis by the COMPARE program.The effect of steric hindrance on reactivity toward anticancer agents seems to be an important parameter in improving the activity.The cytotoxicity results strongly suggest that a fruitful selection of groups with minimum steric hindrance and substituents nature concerning donating and withdrawing effect may be a feasible pathway to derive anticancer agents.
According to the anticancer one dose-response graph, it is justified that most of the compounds have higher GI 50 values against K-562(I), UACC-62(V), CAKI-1(VII), HCT-116(III), NCI-H522(II), MOLT-4(I), RPMI-8226(I), UO-31(VII), PC-3(VIII) cell lines as compared to other given cell lines.The single dose-response of active molecules among synthesized is summarized in Table 3.We divided the effectiveness of compounds into three categories; the first class was for highly active compounds i.e., 5b and 5e, which were shown the consequential effectiveness in leukemia, colon cancer, melanoma, and renal cancer in most cell lines.Compound 5b showed major influence in 'Renal cancer' with the negative value for cell-line, CAKI-1 (GI 50 À4.66).The compound 5b showed other influence of GI 50 value in NSCLC (NCI-H522 ¼ 64.95); one in colon cancer (HCT-116 ¼ 55.73); in Melanoma cancer (UACC-62 ¼ 56.00) and one in prostate cancer (PC-3 ¼ 65.96).On the other hand, compound 5e showed the highest efficacy in MOLT-4 with a À5.37 value in the 'Leukemic cell line'.Furthermore, it shown growth inhibition in UACC-62 ¼ 59.75 (Melanoma cancer), CAKI-1 ¼ 47.05 (Renal cancer), and HCT-116 ¼ 65.17 (Colon cancer).The second category of the compounds was 5c, 5d, 5f and 5j due to moderate activity against some of the cancer panels.These four molecules were found to be comparative active in Leukemia, Colon Cancer, Melanoma cancer and Renal Cancer with the GI 50 values as good as compared to not active molecules against specific cell lines (K-562, MOLT-4, HCT-116, UACC-62, and UO-31 with average inhibition values) in each panel.The third category of the compounds was 5a, 5 g, 5h, and 5i which showed very low/no growth inhibition values.
Out of 10 derived scaffolds, the molecules 5b and 5e contain electron-withdrawing influence from Bromo and Chloro groups at C-2, 3 and/or 5 positions enhancing the effectiveness of the compounds (Figure 4).From the results, it is shown the greater number of halogen sites in the substituents will help to make the molecule more active and the literature survey shows that the Chloro group possesses a special place to enhance biological activity.To explain the differences among the biological activities of these compounds, in this work we pursued to gain insight into the role of the halogen's atom on the chemical reactivity toward cancer cell lines and it was found that the amine ring having Chloro and Bromo functionalities with the number of halogen atoms enhances the activity while those with electron-donating part lower the efficacy in almost all celllines.

Conclusion
In summary, the Ugi four-component coupling processes are catalyzed by catalytic amounts of Au(PPh 3 )OTf.These transitional metal triflates are most likely increasing the yield of this process by activating its imine intermediate.It broadened the use of transition metals in multicomponent reactions.Considering the reality and references mentioned above, as well as the numerous therapeutic applications of the tetrazole molecule in medicinal research.To synthesized substituted tetrazole-based derivatives, we intended to develop an attractive transition metal-catalyzed method based on Ugi's four multicomponent reactions.Furthermore, to take advantage of the most practical use of Metal catalyzed Ugi MCR in terms of higher atom economy and efficient interaction, we synthesized Tetrazole derivatives using the Ugi four-component condensation reaction and attempted the synthesis of a library of unreported substituted tetrazole derivatives and assessed their anticancer activities against the NCI-60 cell line.Overall, compounds 5b and 5e displayed considerable anticancer action against Renal cancer and Leukemia cancer cell line and were shown to be more active than other molecules.The current strategy asks for the use of Ugi-azide chemistry to be broadened in organic synthesis and medicinal chemistry.

Table 1 .
Optimization of Ugi-4CR reaction under precious metal catalytic conditions for the representative molecule (5a).